Analysis of high solid content in biological samples by flame atomic absorption spectrometry by fiona_messe

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                          Analysis of High Solid Content in
                       Biological Samples by Flame Atomic
                                   Absorption Spectrometry
                                                                  Lué-Merú Marcó Parra
               Universidad Centro-Occidental Lisandro Alvarado, Decanato de Agronomía,
                           Dpto. Química y Suelos, Núcleo Tarabana, Cabudare, Edo. Lara
                                                                             Venezuela


1. Introduction
The analysis of organic samples by flame atomic absorption spectrometry (FAAS) involves
the difficulties of the digestion step. This fact was partially overcome by the use of the
microwave assisted digestion technique (Skip, 1998). The digestion of the samples has the
analytical advantage of an appropriated presentation for the analysis by different
techniques. Nevertheless it has some disadvantages as could be analyte losses, risk of
contamination, higher cost, longer analysis time and obligatory dilution of analyte, in some
cases to undetectable levels (Bugallo et al, 2007; Marcó and Hernández, 2004). It is desirable
the development of methods those avoid the sample digestion step. The technique of flow
injection atomic absorption spectrometry is well suited for this purpose (Trojanowicz, 2000),
in the determination of elemental levels in slurry samples and high solid content samples. It
allows to the analysis of solid samples in a simple manner. (Arroyo et al, 2002; Koleva and
Ivanova, 2008).
The analysis of slurry samples gives the advantage of a liquid while allowing the
introduction of a solid. The slurry method is reported for the analysis of prior dried samples
(Januzzi et al, 1997; Da Silva et al, 2006; Mokgalaka et al, 2008), precalcined (Andrade et al,
2008). The analysis by FAAS of solid samples or high solid content samples, as could be
crude clinical samples is not frequently found in the literature. It is reported the use of
slurries from crude tissues combining the FAAS technique with nebulization with a
Babington type nebulizer for the introduction of high solids content samples (Mohamed and
Fry, 1981; Fry and Denton, 1977).
Brandao et al, 2011 reported a simple and fast procedure for the sequential multi-element
determination of Ca and Mg in dairy products employing slurry sampling and high
resolution-continuum source flame atomic absorption spectrometry (HR-CS FAAS). The
main experimental conditions optimized were 2.0 mol L−1 hydrochloric acid, sonication
time of 20 min and sample mass of 1.0 g for a slurry volume of 25 mL. The elements were
determined using        aqueous standards for the external calibration with limits of
quantification of 0.038 and 0.016 mg g−1, respectively. The precision expressed as relative
standard deviation varied from 2.7 to 2.9% for a yogurt sample containing Ca and Mg
concentrations of 1.40 and 0.13 mg g−1, respectively.




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Erik G.P. da Silva et al, 2008 evaluated the slurry sampling flame atomic absorption
spectrometric method for the determination of copper, manganese and iron in oysters
(Crassostrea rhizophora), clams (Anomalocardia brasiliana) and mussels (Mytella guiyanensis;
Perna perna). They optimized the variables nature and concentration of the acid solution for
slurry preparation, sonication time and sample mass. The optimized conditions were 80 mg
of sample grounded in a cryogenic mill, dilution using 1.0 mol L-1 nitric /hydrochloric acid
solution, sonication time of 30 min and a slurry volume of 10 mL. The calibration curves

copper, manganese and iron by FAAS, with detection limits of 0.17, 0.09 and 0.46 g g-1,
were prepared matching the acid concentration. This method allowed the determination of


and 3.8% (n= 10), for concentrations of copper, manganese and iron of 17, 22 and 719 gg-1,
respectively. The precision, expressed as relative standard deviation (RSD), was 3.0%, 2.9%

respectively. The accuracy of the method was confirmed by analysis of the certified oyster
tissue (NIST 1566b). The results showed no significant differences using the proposed
method respect to those obtained after complete digestion and determination by inductively
coupled plasma-optical emission spectroscopy (ICP-OES).
The ultrasonic extraction is an interesting aid to the slurry sampling but its use is not
widely spread as the digestion procedures (Taylor et al, 2002; Taylor et al, 2006). An
ultrasound-assisted solid–liquid extraction procedure by using diluted mixed acid solution
was developed by Manutsewee, et al, 2007 for determination of cadmium, copper and zinc
in fish and mussel samples. They evaluated the effects of several parameters such as nitric
acid concentration, hydrochloric acid concentration, hydrogen peroxide concentration,
leaching solution volume, temperature and sonication time. After the optimization of these
parameters the elements cadmium and copper were determined by graphite furnace atomic
absorption spectrometry, and zinc was determined by flame atomic absorption
spectrometry. The results were compared to those obtained by microwave-assisted
digestion. The recoveries (%) of metal amount obtained by leaching technique to the amount
obtained by digestion technique for cadmium, copper and zinc ranged from 92% to 114% for
fish and from 88% to 103% for mussel samples. The accuracy of the developed method was
verified with the dogfish muscle certified reference material (DORM-2). The Recoveries
were in the order of 80.9 ± 0.3 and 87.2 ± 0.6%.
Bugallo et al, 2007 compared the method of slurry sampling to the microwave assisted
digestion for the determination of calcium, copper, iron, magnesium and zinc in fish tissue
samples by flame atomic absorption spectrometry. They found that in comparison to
microwave-assisted digestion, the analysis of slurries is simple, requires short time and
overcome the difficulty of the total sample dissolution before analysis. It is necessary the
addition of acid for some analites as iron, to enhance the recovery. For Ca and Cu the
quantification must be performed using standard addition. Both methods were accurate and
the standard deviations obtained using slurry sampling method and microwave-assisted
digestion were not significantly different and the mean relative standard deviation of the
slurry sampling method for different concentration levels was below 12%.
In this chapter will be discussed the analysis by FAAS of two kinds of biological samples:
brain and onion bulb tissues. The preparation procedures were simplified in order to
perform the analysis of the crude samples with the aid of ultrasonic acid extraction for brain
slurries and crude onion leachates. The introduction using a Flow Injection System avoids
transport effects. The results by FAAS and flow injection atomic absorption spectrometry
(FIAAS) were evaluated using the independent technique of TXRF.




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Analysis of High Solid Content in Biological Samples by Flame Atomic Absorption Spectrometry   185

1.1 Determination of Zn and Cu in crude human brain slurry samples by flow injection
flame atomic absorption spectrometry
The determination of metals in brain samples is required for the study of brain
physiology, biochemistry and neurochemistry, or clinical purposes to find correlations
between metal levels and some pathologies (Schizophrenia, Wilson disease, Alzheimer,
etc.) (Andrási et al, 1995; Religa et al, 2006). In connection to neurochemistry, it is
necessary to determine the elements Zn and Cu for the evaluation of neurotransmission
processes. (Horning et al, 2000).
The brain matrix is mainly formed by high molecular weight carboxilic acids or greases of
long chains, a complex organic matrix. The digestion of such a matrix prior to the analysis is
reported in the literature, for different techniques (Andrade et al, 2008; Taylor et al, 2002;
Taylor et al, 2006; Lech and Lachowicz, 2009). The main objective of this work was the
determination of Cu and Zn in crude brain slurry samples by the method of flow injection
atomic absorption spectrometry and the development of precise, accurate, efficient and
cheap method of determination of metals in human brain samples. The results were
compared to those obtained after microwave aided digestion of the samples. The
independent technique of total reflection X-ray fluorescence (TXRF) was used for accuracy
evaluation.
Crude brain dissected samples were homogeneized with a high speed homogenator to
obtain slurries. These slurry samples were properly diluted in 5% V/V HNO3 to aid the
analyte extraction to the aqueous phase, to carry out the determination of copper and zinc
by flame atomic absorption spectrometry, following a procedure reported by Marco et al,
2003.

1.2 Determination of calcium, potassium, manganese, iron, copper and zinc levels in
representative samples of two onion cultivars by flame atomic absorption
spectrometry using ultrasonic assisted leaching
The onion is one of the most important cultivars in the world. The determination of major,
minor and trace element levels is an important tool for the enhancement of production
efficiency in the field of agriculture, provenance, and contamination risk evaluation (Ariyama
et al, 2007; Abdullahi et al, 2008; Abdullahi et al, 2009). There is a necessity for new analysis
methods and simple sample preparation procedures. The chemical characterization of the
cultivar samples, becomes important due to the fact that chemical composition is closed
related to the quality of the products. (Akan et al, 2010; Hashmi et al, 2007). Alvarez et al, 2003,
reported a preparation procedure for the elemental characterization, involving the acid
extraction of the analytes from crude samples by means of an ultrasonic bath, avoiding the
required digestion of samples in vegetable tissue analysis. The technique of total reflection X-
ray fluorescence (TXRF) was successfully applied for the simultaneous determination of the
elements Ca, K, Mn, Fe, Cu and Zn. The procedure was compared with the wet ash and dry
ash procedures for all the elements using multivariate analysis and the Scheffe test. The
technique of flame atomic absorption spectrometry (FAAS) was employed for comparison
purposes and accuracy evaluation of the proposed analysis method. A good agreement
between the two techniques was found when using the dry ash and ultrasound leaching
procedures. The levels of each element found for representative samples of two onion cultivars
(Yellow Granex PRR 502 and 438 Granex) were also compared by the same method.
In this work a sample preparation procedure for onion bulb elemental characterization by
FAAS is proposed, involving the acid extraction of the analytes by means of an ultrasonic




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186                                                             Atomic Absorption Spectroscopy

bath. The procedure was compared with the wet ash and dry ash procedures for all the
elements using multivariate analysis and the Scheffé Test. The accuracy was also verified
with the TXRF technique. The onion samples were grounded and homogenized with
deionized water (1:1) in a domestic homogenator. In a second step, nitric acid was added at
different concentrations for the optimization of acid levels. The samples were placed for 30
minutes in the ultrasonic bath. It was found an optimal concentration of 5% V/V of nitric
acid and 10% of wet sample mass. Then samples were filtrated. The filtrates were also

elements, using deionized water as carrier, an optimized injection volume of 150 L and an
analyzed by flow injection flame atomic absorption spectrometry (FI-AAS) for all the

optimized flow of 3.5 ml/min. The results obtained were compared to those obtained using
the methods of wet ash and dry ash sample digestion and flame atomic absorption analysis
after the humidity correction (dry base). For two kind of cultivars (Yellow Granex PRR 502
and 438 Granex). it was found that the dry ash method was statistical equal to the method of
ultrasonic extraction-FIAAS. No significant differences were found between the results
obtained by FAAS and TXRF. The precision was always below 5% of relative standard
deviation in all the cases. It was concluded that the proposed method is the most reliable in
the basis of its simplicity, shorter analysis time and minor use of reagents and glassware.

2. Experimental
2.1 Analysis of brain samples
2.1.1 Samples
Brain samples from healthy, male individuals, who suffered accidental and/or
instantaneous death were taken at Morgue of the Central Hospital of Barquisimeto, Edo.
Lara, Venezuela. Brains were dissected, not more than 24 hours after death, and kept at -50
oC until sample preparation.



2.1.2 Sample treatment
The brain sections, such as cerebellum, hypothalamus, frontal cortex and encephalic
trunque, were weighed and homogenised with deionized water with a high speed
homogeneiser at 23000 rpm (Ultra-Turrax P25 Janke and Kumkel, IKA registered mark-
LABORTECHNIK). Homogenates with a 50-60% w/V (wet weight) of brain tissue were
obtained and kept at -20 oC until analysis. Some samples were lyophilized after
homogenization at –50 oC in a digital LABCONCO lyophiliser, LYPH-LOCK.
The digestion of the homogenates and lyophilized samples for comparison purposes was
carried out in a Domestic microwave oven using closed teflon vessels, in two steps: 15
minutes at medium power and 10 minutes at maximum power. An ultrasonic bath Cole
Palmer was employed for slurry treatment and for homogenization. The whole sample
treatment is detailed by Marcó and Hernández, 2004.

2.1.3 Slurries
Slurries were prepared taking aliquots of the 50-60% w/V crude brain homogenates with
volumetric pipettes and transferring to calibrated flasks, following strictly the next
procedure:
1. An appropriate aliquot of homogenate is taken with the glass volumetric pipette,
     depending on the desired concentration of the slurry .




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Analysis of High Solid Content in Biological Samples by Flame Atomic Absorption Spectrometry   187

2.    The homogenate portion is then transferred with the pipette to the volumetric flask. In
      this step, the pipette is rinsed with deionised water helping the remaining homogenate
      in the inner wall of the pipette falling to the volumetric flask.
3. Deionized water is added to the sample into the flask to fill approximately half of its
      volumetric capacity and flask was slightly agitated for few seconds to form an
      homogeneous slurry.
4. In the case of acid addition, the appropriate amount of the nitric acid is added to the
      flask, depending of the desired acid concentration.
5. The calibrated flask with the slurry is finally filled to the labelled volume, with
      deionised water.
6. The sample slurries in the flask are treated for 10 minutes in ultrasonic bath at 25 oC.
It is important to remark that this procedure must be followed in all the cases, in order to get
stable, and homogeneous slurry. When the step order is not followed, it is frequently to
observe the instantaneous denaturalization of the homogenate and the formation of particles
of non desirable size.
Additional reagents for assurance of the slurry stability, such as Viscalex, Triton among
others were not necessary. Problems with foaming were not found.

2.1.4 Digested samples
In a similar way as described in steps 1 and 2 of the slurry preparation procedure, a sample
aliquot of 5 ml of homogenate was transferred to the teflon vessels, instead of the volumetric
flasks. Then 5 ml of ultrapure concentrated nitric acid and drops of hydrogen peroxide were
added to the teflon vessels. Vessels were closed for digestion in the microwave oven.
About 0.35 g of Lyophilised brain samples were weighed and digested in closed teflon
vessels with 5 ml. of concentrated nitric acid and drops of hydrogen peroxide using the
same microwave assisted digestion followed with the brain homogenates.

2.1.5 Standards and reagents

stock solution (1000 g mL-1), Titrisol, Merk. Zinc determination was carried out using
Aqueous calibration standards of Zinc and Copper were prepared by serial dilution of the

always aqueous calibration curves. Copper determination was carried out using the
aqueous calibration curve and also the standard addition method. Suprapur, 65% v/v
HNO3 (MERCK, Germany) and 30% v/v H2O2 (Riedel de Haen, Germany) were employed
for leaching and digestion purposes. Nitric acid (Riedel de Haen, Germany) was used for
cleaning quartz reflectors for the TXRF analysis. Distilled, deionized water (16 MVcm ) was
employed for rinsing and dilution purposes and also as FIAAS carrier.

2.1.6 FAAS analysis
Measurements were performed in a 2100 Perkin Elmer flame atomic absorption
spectrometer. FIAAS set up without peristaltic pump, using the nebulizer aspiration flow
for sample and carrier propulsion as shown in figure 1 was used to avoid the clogging of the
system. This system was made of Teflon pieces from a chromatographic column kit. In one
position of the valves the loop is charged while the carrier is passing direct to the nebulizer.
In the other position of the valves, the sample in the loop is aspirated and the carrier
immediately passes through the loop to the nebulizer. As the system has the possibility of
two loops, when one of the loops is being charged the carrier is passing through the other




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loop and viceversa. The carrier container is an extraction funnel fixed 30 cm above the entry
of nebulizer. The volume of the sample loop was approximately 100 microliters. The length
of the tube from injection port to nebuliser was 35 cm and the internal diameter 0.8 mm.




Fig. 1. Sample introduction system
TXRF measurements
A CANBERRA Energy Dispersive X-Ray Spectrometer, with Si-Li detector and set up for
TXRF, Excitation with Mo tube, 17.4 Kev line was used for X-Ray analysis. TXRF set up with
TLAP crystal as monochromator. Details of the TXRF measurements are given in the works
of Marcó et al, 1999 and Marcó and Hernández, 2004.

2.2 Analysis of onion samples. (According to Álvarez et al, 2003).
2.2.1 Reagents and standards
Titrisol, 1000 standard solutions (MERCK, Germany) were employed for preparation of
calibration curves. Suprapur, 65% v/v HNO3 (MERCK, Germany) and 30% v/v H2O2
(Riedel de Haen, Germany) were employed for leaching and digestion purposes. Nitric acid
(Riedel de Haen, Germany) was used for cleaning quartz reflectors for the TXRF analysis.
Distilled, deionized water (16 MVcm ) was employed for rinsing and dilution purposes and
also as FIAAS carrier.
The standards for FAAS and FIAAS analysis were prepared by serial dilution of titrisol
standards, according to the linear range of each of the analyzed elements, as reported by the
manufacturer.
For the TXRF analysis aqueous, multielement (K, Ca, V, Mn, Cu, Se and Sr) standards were
prepared by mixing and dilution of the corresponding stock solutions with distilled, de-




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Analysis of High Solid Content in Biological Samples by Flame Atomic Absorption Spectrometry   189

ionized water to yield final concentrations of 5, 10 and 20 mgL-1. The element Co was used
as internal standard and added to standards and samples. The further quantification is well
explained by Alvarez et al, 2003.

2.2.2 Sampling procedure
Two kinds of onion cultivars were collected, Yellow Granex PRR of Sumblex and Texas
Grano 438, Asgrow, at the main market of Barquisimeto following the next procedure: ten
bags of 60 Kg from each cultivar were random selected. Then from each bag were taken 10
onions. A total amount of 100 onions for each cultivar were collected.
Sample preparation
The onion samples were grounded and homogenized with deionized water (1:1) in a
domestic homogenizator in a previous step (30 random selected onions for each preparation
procedure from each cultivar) . An amount of 5 g of wet weight or 10 g of the homogenate
was digested by a wet ash procedure and prepared by ultrasonic leaching, as explained
bellow. In similar way an amount of dry onion corresponding to 5 g of wet weight
(calculated on the basis of the dry matter content) was used for the dry ash procedure. The
humidity correction and the determination of dry masses were performed separately by
drying in oven at a fixed temperature of 60 oC. The values of dry matter percentage were 8%
for Yellow Granex and 9% for 438 Granex cultivar. The results are expressed in dry basis. In
all the cases four independent replicas were prepared.

2.2.3 Ultrasonic extraction
An ultrasonic bath, Cole Palmer (USA) with temperature control was used. Temperature
was fixed to 70 oC. Time of sonication was 30 min. Ten grams of the homogenate (5 g of wet
sample) were placed in flasks and mixed with different nitric acid concentrations (0, 5, 10
and 15% v/v) for the optimization of acid levels. The samples were placed for 30 min in the
ultrasonic bath with a fixed temperature of 70 oC. Then samples were filtrated with
Whatman filters by gravity and the supernatants were quantitatively transferred to 50 mL
volumetric flasks.

2.2.4 Wet ash
The wet digestion was performed weighing 5 g of the wet sample and adding 15mL of
concentrated HNO3 and drops of H2O2. The digestion was performed in a hot plate. After
the digestion procedure the sample were aphorized to a final volume of 50 mL.

2.2.5 Dry ash
The dry digestion was performed weighing 0.4 g of dry sample (approx. 5 g of wet sample)
and calcining at 700 oC for 2 h. Then, the ashes were dissolved with nitric acid and samples
were quantitatively transferred to 50 mL volumetric flasks.

2.2.6 FAAS analysis
The samples were analyzed in a Perkin Elmer (USA) 3110 Atomic Absorption Spectrometer
under conditions suggested by the manufacturer.
The FIAAS manifold was designed for low dispersion and two channels ( carrier and
sample), using a An ISMATEC peristaltic pump IPC model for sample and carrier




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190                                                              Atomic Absorption Spectroscopy

introduction controlled by a Temporizer GrabLab model 900 and a control valve Cole-
Palmer, model 625E Bunker CT. See Figure 2.




Fig. 2. FIAAS manifold

2.2.7 TXRF analysis
The TXRF analysis was carried out in a Canberra XRF Spectrometer with a modified TXRF
module designed at the Atominstitut der Osterreichschen Universitaeten, Vienna. The
excitation was performed with the Ka (17.4 keV) line of a molybdenum anode X-ray tube,
operated at 40 kV and 20 mA, as detailed by Alvarez et al, 2003. A carbon -molybdenum
multilayer crystal was used for monochromation of the incident beam and a Si(Li) detector
(Resolution 180 eV at Mn line, 5.8 keV) was used for the detection of the fluorescence signal.
The spectra were collected in a PC based multichannel analyzer (Canberra S100), with live
collection time of 200 s. The spectral data analysis was conducted with the AXIL fitting
program and QXAS package supplied by the International Atomic Energy Agency.

3. Results
3.1 Analysis of brain samples
The feasibility of the crude brain slurry direct introduction was tested at a first stage using
different concentration (w/V) of brain tissue in water with and without acid. The
introduction of the samples was performed by the use of a simple flow injection system
described in the figure 1 due to frequent obstruction of the valve of the FIAAS manifold
with peristaltic pump. Optimal slurry concentration was in the range of 2.3% w/V to 12.5%
w/V for zinc determination while for copper the slurry concentration must be higher than
20% w/V for an appropriate detection and less than 24% w/V to avoid matrix interferences.
The results obtained by the slurry method were compared to those obtained after sample
digestion and also to the independent technique total reflection X-ray fluorescence. A good
agreement between results confirmed the accuracy of the proposed sample preparation
procedure. The mean precision for the zinc and copper determination was less than 5% for
most of the samples.

3.1.1 Optimization of experimental conditions
Experimental conditions for the FAAS method were fixed following the routine recommended
by the spectrometer manual. The parameters as slit, lamp current and gas flow were




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Analysis of High Solid Content in Biological Samples by Flame Atomic Absorption Spectrometry   191

automatically selected. For the FIAAS method, variations of the gas flow were performed. Gas
flow was changed from 5 L/min to 8 L/min. A slight increment in sensitivity was observed
when using 5L/min of gas flow, nevertheless the obstruction of the capillary tube was
frequently observed under this condition due to the high solid content of the brain slurry
samples. High gas flow was necessary to avoid the capillary tube obstruction.
Sample volume: three sample loop sizes were tested: 100, 150 and 200 microliters. The 100
microliters loop was selected as optimal volume, since no significant changes are observed
in peak height respect to higher volumes, dispersion is low and risk of memory effect are
minimized. The dispersion was 1.3 for Cu and Zn in the optimized set up.
slurry concentration and stability: Slurries with concentration w/V of 2.3%, 12.5% and 23 %
were tested. Simultaneously, the effect of nitric acid 5% w/V was evaluated. The slurry 2.3%
corresponded to the minimal concentration that allowed to the measurement of Zinc signal
in the lower value of the working calibration range. The 12.5% is a concentration in the
range recommended by Mohamed and Fry, 1981 for homogeneized tissues and 23.5%
corresponds to the critical matrix due to the high solid content. The stability of the
absorbance signal does not depend on the acid at concentration levels lower than 12.5%,
evidencing that the analyte is mostly in the aqueous phase. It was deduced that for Zn a

Concentrations higher than 13% are over the linear calibration range (0.1-1 gml-1). In the
slurry concentration less than 12.5 % w/V and 5% HNO3 V/V is adequate for the analysis.


since linear range lies at higher concentration values, between 1-10 gml-1. The critical
case of copper, slurry concentration must be higher than 12.5% for an appropriate detection

higher concentration was 24%, due to matrix effects. See figure 3.




Fig. 3. Effect of the Slurry Concentration on Zn (■) and Cu(○) Signals.




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Effect of nitric acid: the effect of nitric acid was evaluated at concentrations of 5% w/V, 10%
w/V and 20% w/V. An increment of the absorbance signal is observed when the acid is
added. The quality of the slurries in terms of particle size is improved. The clogging of the
capillary was observed for samples without acid. Acid concentrations above 5% v/V do not
affect significantly the absorbance signal. The acid blank was tested and its influence
discarded on the measurement.

3.1.2 Determination of Zn
The determination of Zn can be carry out by slurry sampling at slurry concentration levels
(%w/V) between 2.3 and 13% if nitric acid is added. Nevertheless, for concentration levels
near the lower limit (2.3%) a high relative standard deviation is obtained. See table 1.


                     SAMPLE       CONCENTRATION              RSD (n=5)

                     Ce-2              8.8 (12%)                 3%
                                        8.5 (4.8%)                4%
                     Te-2              7.7 (12%)                 2%
                     Te-1              5.6 (4.8%)                3%
                     Cf-1              9.6 (4.8%)                2%
                     Ce-1              11.0 (4.8 %)              2%
                     Hp-1              10.9 (4.8%)               3%
                     Te-3              14.2 (2.3%)               9%
                     Hp-3              10.0 (2.7%)               10%
                     Cf-3              5.6 (4.7%)                 5%

Table 1. Concentration (g g-1) of Zn in Crude Brain Slurry Samples. The values in
parenthesis indicate the slurry concentration. RDS: Relative Standard Deviation. Ce:
Cerebellum; Te: Encephalic trunque; Hp: Hypotalamus; Cf: Frontal Cortex.
A good concordance between results obtained by slurry sampling and those obtained after
microwave digestion of crude brain samples was observed, as shown in table 2. If the results
for crude samples are compared to the corresponding liophylized and digested samples,
the analyte concentration is in concordance to the dry weight correction (about 20% in brain
samples ( Andrási et al, 1995 ). The test of the correlation factor F at the 0.05 level of
confidence was applied ( F = 120.7 p= 0.002 and critical F 10.1) demonstrating the agreement
between results. See table 2.
The precision ranges between 2% of relative standard deviation (RSD) and 10%. This
parameter is independent of the sample preparation procedure (slurry or digested) or the
brain section. The precision depends on the analyte concentration. For slurry concentration
above 4.8% w/V the value is under 5% of RSD. The precision when comparing independent
replicates was similar.




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The accuracy was evaluated by comparison to the technique of TXRF for the slurry and
digested samples (see table 3). The t-Student at 95% of confidence level was applied (t= 0
and p=1).

 Sample             Crude                         Digested          Lyophilised and Digested
 CE-2               8.8 (3%)                      9.0 ( 2%)
 Et-1               5.6 (3%)                      5.1 (4%)                    5x5.4
                                                                              27 (3%)
 CE-1               11.0 (2%)                     10.5 ( 5%)                  5x13.2
                                                                              66 ( 2%)
 Hp-1               10.9 ( 3%)                    9.9 ( 2%)
 Et-3               14.2 (9%)                     12.9 ( 3%)
Table 2. Comparative Results of Zn Concentration (gg-1) in Crude Brain Slurries and
Digested Brain Samples. The values in parenthesis correspond to relative standard
deviation.


                 Sample              TXRF                        FIAAS
                 Cf1                 2.3 +/-0.2
                                   **2.1 +/-0.2                  2.3 *(3%)
                 Ce1                 2.3 +/-0.2                  2.6 (2%)
                                  **2.4 +/-0.2                 **2.5 (5%)
                 Cf2                 2.2 +/-0.3                  2.1 (1%)
                                                               **2.2 (2%)
                 Ce2                 2.2+/-0.3                   2,0 (2%)
Table 3. Comparative Results of Zn Concentration (gg-1) Obtained by TXRF of Slurries,
TXRF of Digested Samples , FIAAS of Slurries and FAAS of Digested Samples. The values in
parenthesis indicate the relative standard deviation. ** The value corresponds to digested
sample. Cf: Frontal Cortex; Ce: Cerebellum

3.1.3 Determination of Copper
The slurries for Copper determination should have a concentration higher than 20% w/V,
due to the low concentration of the analyte in the samples. Slurries with concentration less

calibration range (1 g mL-1). Values of slurry concentration higher than 24% (w/V) have
than 20% (w/V) have an analyte concentration bellow the lowest point of the working

the lack of matrix effects, transport effects and capillary tube obstruction among others. The
nitric acid should be added to the slurries in order to extract efficiently the analyte to the
aqueous phase and to enhance the nebulisation and atomization processes in the spray
chamber and into the flame. See table 4.
The copper determination was performed by direct FIAAS analysis and by the standard
addition method (see reference 78), due to the high concentration (w/V) of the slurries. As
shown in table 4 no significant differences were found between results using standard
addition method and the direct method. The correlation coefficient between results was
0.994 the slope 0.99 and intercept 0.017. The t-Student test at 95% confidence level was t=




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0.11, p= 0.92. These results demonstrate that even for slurries at 24% w/V no matrix effects
are observed. This fact is a consequence of the addition of the nitric acid and the analyte
extraction to the aqueous phase.
The precision of the results for the FIAAS-Slurry method was between 3% of RSD and 11%
of RSD. Nevertheless precision values higher than 5% RSD were not observed for digested
samples. Then the parameter is affected by the matrix.
The accuracy was evaluated by comparison to the technique of TXRF, as shown in table 5.
When FIAAS method is compared to TXRF using the t-Student test at 95% of confidence
level (t=0; p=1), demonstrating the good agreement between the results obtained by both
techniques.

                Sample            Cu Concentration gg-1 (%RSD)
                            Standard Addition           Calibration Curve
                Ce1               0.93*(10%)                0.92(4%)
                Ce3               0.68(6%)                  0.71(4%)
                Te5               0.52(6%)                  0.52(7%)
                Cf1               0.78(3%)                  0.80(11%)
                Ce4               0.98(5%)                  0.94(10%)
                Ce2               0.94(3%)                  0.92(4%)
                Cf4               0.72(2%)                  0.71(3%)
                Cf3               0.72(5%)                  0.62(3%)
                P1                1.55(3%)                  1.50(3%)
                P2                1.58(2%)                  1.59(1%)
Table 4. Concentration of Cu (gg-1) in Crude Brain Slurry Samples Determined Using
Standard Calibration Curve vs. Standard Addition Method of Determination. The values in
parenthesis indicate relative standard deviation.



                                    Concentration of Cu (gg-1)
                  Sample            TXRF             FIAAS
                  Cf1               0.8 +/-0,1       0.77*(3%)
                  Cf4               0.8 +/-0,1       0.72 (2%)
                  Ce3               0.9 +/-0,1       0.68 (6%)
                  Cf2               0.6 +/-0,1       0.69 (9%)
                  Ce1               1.1 +/-0,1       0.93*(10%)
                  Te3               0.5 +/-0,1       0.52 (6%)
                  Ce2               1.0 +/-0,1       0.93 (3%)
                  Te2               0,6 +/-0,1       0.56 (6%)
Table 5. Copper Levels in Crude Brain Slurries by Standard Addition-FIAAS Method vs.
TXRF. The values in parenthesis indicate relative standard deviation.




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Analysis of High Solid Content in Biological Samples by Flame Atomic Absorption Spectrometry   195

3.2 Analysis of onion samples
3.2.1 Optimization of experimental conditions
Recovery efficiency in acid medium: It is expected that the partial extraction of the analite to the
supernatant occurs when acid is added to a slurry. The atomization efficiency is enhanced,
in addition due to the fact that the acid helps to decrease the particle size.
Taking this fact into account, diluted nitric acid was employed at concentration levels of 0, 5,
10 y 15% v/v, in water. The signal increases due to the HNO3 effect, being the optimal value
5%, with 100% of recovery for the determined elements. A slight suppression of the signal
was observed for HNO3 (10 y 15%) and in consequence lower % recovery for Mn, Zn and Fe.
The addition of HNO3, induces a predigestion of the solid phase, decrement of particle size
and almost the total extraction of the analites into the aqueous phase. (See table 6). The
elements Ca and K are extracted to the aqueous phase with deionized water.

 Element      % of recovery
              0 % V/V HNO3           5 % V/V HNO3        10 % V/V HNO3          15 % V/V HNO3
 Fe           30                     101                 103                    104
 Mn           34                     100                 98                     96
 Zn           33                     102                 80                     85
 Cu           25                     98                  94                     93
 K                                                     100
 Ca                                                    100
Table 6. Percent of recovery in onion bulb samples after ultrasonic extraction procedure as
function of the nitric acid concentration (% V/V).
Optimization of FIAAS parameters: the optimized FIAAS parameters were pump flow rate (3.5
mL/min), suction flow rate set 0.2 units bellow pump flow rate (3.3 mL/min). As it is
deduced from table 7 there are not significant differences in the recovery, but the highest
rate tested ensures a minor residence time of the sample in the nebuliser and chamber, and
in consequence a lesser memory effect. The optimal sample volume was fixed at 300 l. It
was found that sample volumes higher than 350 l did not allow to a significant
enhancement. In this case there is not compensation of the matrix effect by dispersion and
the signal was similar to that of the classical FAAS analysis. See Figures 4 and 5.


 Element             % of recovery
                        2 mL/min             2.5 mL/min            3 mL/min          3.5 mL/min
 Fe                             86                   89                  88                  89
 Mn                             92                   93                  93                  93
 Cu                             97                   98                  97                  99
 Zn                            100                  101                 101                 103
 Ca                                                    100
 K                                                     100
Table 7. Percent of recovery for Fe, Mn, Cu, Zn, Ca and K as function of the pump flow rate

volume 350 L.
in the analysis of onion bulb leachates by FIAAS. Nitric acid (5% v/V) and sample loop




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196                                                              Atomic Absorption Spectroscopy




Fig. 4. Relative Absorbance signal as function of the injected sample volume and nitric acid
concentration. 5% V/V (♦), 10 % V/V (■) and 15% v/v (▲).




Fig. 5. Relative absorbance as function of the injected volume for leachates of the onion
varieties Yellow Granex (YG) and Texas Grano 438 (GR). Fresh sample mass 5 g in a volume
of 50 mL. Pump flow rate 3.5 mL/min.




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Analysis of High Solid Content in Biological Samples by Flame Atomic Absorption Spectrometry        197

3.2.2 Comparison of the results obtained by the preparation procedures using FAAS
and the FIAAS method
It was found that in general, the dry ash procedure and the FIAAS method had not
significant differences when the Sheffe test was applied in the comparison of the results for
all the elements (See table 8). The results are higher than those of the wet ash procedure and
the direct aspiration of the leachates by FAAS, with the exception of Potassium. This trend is
the consequence of the stronger matrix effect in these procedures. In the wet ash treatment
the simple mineralization could not be total as occurs with the wet ash. In the case of the
direct aspiration of the leachates by FAAS, the particle size is higher and the matrix was not
totally eliminated. The nebulisation and atomization processes were affected in a different
way as the aqueous standards used for calibration. The FIAAS procedure and the
subsequent dispersion reduced the matrix effects. The addition of nitric acid allowed to the
extraction of the analites to the aqueous phase with the increment in the nebulisation and
atomization efficiency as compared to the procedure when the leachate is directly aspirated
by FAAS.

                                             Preparation Procedure
                                                          Ultrasonic Leaching- Ultrasonic Leaching-
                Dry Ash               Wet Ash                    FAAS                 FIAAS
Element      YG           G         YG            G          YG          G         YG           G
 Ca %     0.400*(0.6) 0.310 (1) 0.38(0.6) 0.30 (3) 0.37 (0.9) 0.30 (1)           0.400 (1)   0.34 (2)


  K%       1.03 (1)   1.38(0.5) 1.06(0.6) 1.80(0.7) 1.07 (0.2) 1.53 (0.5) 1.00 (2)           1.40 (2)


Fe g/g     27 (3)     45 (3)     28.2 (4)     41 (5)       21 (3)    39 (3)     20.0 (3)     40 (3)


Zn g/g 16.7 (3)     19.3 (3)    15.2 (2)     17.7 (3)    11.7 (3)   15.4 (3)   17.3 (4)    19.5 (3)



 g/g
  Mn
           17.1 (2)    40 (4)     12.9 (5)     34 (3)      14.1 (5)    36(4)     15.2 (4)     39 (4)
Table 8. Comparison of elemental concentrations in onion bulb samples using different
methods by Flame Atomic Absorption Spectrometry. N=4. In parenthesis Relative Standard
Deviation. YG: Yellow granex cultivar and G: Texas Grano 438.

3.2.3 Comparison to the TXRF technique
A good agreement was found between the results obtained by FIAAS and TXRF (See Figure
6). The results obtained by FAAS when the leachates are directly aspired, were significantly
lower (p=0.05) with the exception of the element potassium, in the same way as in the wet
ash and dry ash procedures, as explained before. It is important to point that the TXRF
technique has not the matrix effects as the FAAS technique. The agreement between FIAAS
and TXRF demonstrates the effectiveness of the proposed procedure of ultrasonic leaching
and FIAAS analysis for the reduction of the matrix effects and its reliability in the analysis
of onion bulb samples.




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198                                                              Atomic Absorption Spectroscopy




Fig. 6. Comparison of the results by FAAS and FIAAS to the TXRF technique for the
leachates of onion bulb samples using ultrasonic aided extraction.

4. Conclusions
A simplified sample preparation procedure was evaluated for the analysis of human brain
samples. The feasibility of the preparation of crude brain slurry samples for Atomic
absorption spectrometry analysis, in terms of precision and accuracy was demonstrated.
These slurry samples must be properly diluted in 5% V/V HNO3 to aid the analyte
extraction to the aqueous phase, to carry out the determination of copper and zinc by flame
atomic absorption spectrometry. Optimal slurry concentration was in the range of 2.3% w/V
to 12.5% w/V for zinc determination while for copper the slurry concentration must be
higher than 20% w/V for an appropriate detection and less than 24% w/V to avoid matrix
interferences. A good agreement between results by TXRF and FIAAS confirmed the
accuracy of the proposed sample preparation procedure. The mean precision for the zinc
and copper determination was less than 5% for most of the samples.
The determination of K, Ca, Mn, Fe, and Zn in fresh onion bulb samples using ultrasonic
extraction is a reliable procedure when a FIAAS set up is used. The process must be aided with
nitric acid at a concentration level of 5% v/V with five g of homogenized fresh sample in 50
mL. It was demonstrated the substantial reduction of matrix effects if a FIAAS method is
applied. The procedure is fast, simple and has lower cost compared to the wet ash and dry ash
procedures. The accuracy was verified by comparison to the independent technique TXRF and
demonstrated by the good agreement found. The precision for the ultrasonic extraction and
FIAAS set up is less than 5% of relative standard deviation for all analyzed elements.

5. Acknowledgments
The author thanks to the CDCHT-UCLA for the financial support of this research with the
project AG-040-2000.




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Analysis of High Solid Content in Biological Samples by Flame Atomic Absorption Spectrometry   199

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                                      Atomic Absorption Spectroscopy
                                      Edited by Dr. Muhammad Akhyar Farrukh




                                      ISBN 978-953-307-817-5
                                      Hard cover, 258 pages
                                      Publisher InTech
                                      Published online 20, January, 2012
                                      Published in print edition January, 2012


Atomic Absorption Spectroscopy is an analytical technique used for the qualitative and quantitative
determination of the elements present in different samples like food, nanomaterials, biomaterials, forensics,
and industrial wastes. The main aim of this book is to cover all major topics which are required to equip
scholars with the recent advancement in this field. The book is divided into 12 chapters with an emphasis on
specific topics. The first two chapters introduce the reader to the subject, it's history, basic principles,
instrumentation and sample preparation. Chapter 3 deals with the elemental profiling, functions, biochemistry
and potential toxicity of metals, along with comparative techniques. Chapter 4 discusses the importance of
sample preparation techniques with the focus on microextraction techniques. Keeping in view the importance
of nanomaterials and refractory materials, chapters 5 and 6 highlight the ways to characterize these materials
by using AAS. The interference effects between elements are explained in chapter 7. The characterizations of
metals in food and biological samples have been given in chapters 8-11. Chapter 12 examines carbon capture
and mineral storage with the analysis of metal contents.



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Lué-Merú Marcó Parra (2012). Analysis of High Solid Content in Biological Samples by Flame Atomic
Absorption Spectrometry, Atomic Absorption Spectroscopy, Dr. Muhammad Akhyar Farrukh (Ed.), ISBN: 978-
953-307-817-5, InTech, Available from: http://www.intechopen.com/books/atomic-absorption-
spectroscopy/analysis-of-high-solid-content-in-biological-samples-by-flame-atomic-absorption-spectrometry




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